Chondroitin Sulphate (hereafter CS), belonging to the class of natural complex polysaccharides named glycosaminoglycans (GAGs), is composed of alternate disaccharide sequences of differently sulphated residues of D-glucuronic acid (GlcA) and of N-acetyl-D-galactosamine (GalNAc) linked by beta(1→3).
Depending on the disaccharide nature, CS with different carbohydrate backbones are known. In fact, even if both natural and synthetic known CS are mainly composed of various percentages of two kinds of disaccharide units, i.e., sulphated in position 4 or 6 of GalNAc, disaccharides with a different number and position of sulphate groups can be located, in various percentages, within the polysaccharide chains. For example, the unsulphated disaccharide is present, generally in low amounts, in the CS backbone, while disulphated disaccharides having two sulphate groups O-linked in various positions, such as position 2 of GlcA and position 6 of GalNAc (disaccharide D), or in position 4 and 6 of GalNAc (disaccharide E), may be present in the CS backbone in various percentages in relation to specific animal sources [Volpi, N., J Pharm Pharmacol 61, 1271, 2009 and Volpi, N., J Pharm Sci 96, 3168, 2007].
CS shows a disaccharide repeating unit having the following structural formula:
wherein R2, R4 and R6 are independently either H or SO3−.
The meaning of some of the most recurring acronyms, currently used to briefly identify the differently sulphated residues of the alternate disaccharide sequences which make CS, are below reported.
Di-0S(R2 = H; R4 = H; R6 = H)Di-6S (C)(R2 = H; R4 = H; R6 = SO3−)Di-4S (A)(R2 = H; R4 = SO3−; R6 = H)Di-4,6diS (E)(R2 = H; R4 = SO3−; R6 = SO3−)Di-2,6diS (D)(R2 = SO3−; R4 = H; R6 = SO3−)Di-2,4diS (B)(R2 = SO3−; R4 = SO3−; R6 = H)Di-2,4,6triS(R2 = SO3−; R4 = SO3−; R6 = SO3−)
Both natural and synthetic CS samples may be characterized and differentiated by means of sensitive, specific, validated and published analytical approaches, able to give CS structural characterization and parameters (for example specific sulphated groups, charge density, molecular mass, and purity) as well as biological activities.
Natural extractive CS samples may be characterized for structure and properties [Volpi, N., J Pharm Pharmacol 61, 1271, 2009; Volpi, N., J Pharm Sci 96, 3168, 2007; Mucci, A. et al., Carbohydr Polymers 41, 37, 2000; and Volpi, N., Analyt Biochem 277, 19, 2000].
As to the tri- and tetra-sulphated forms of CS (“triS” and “tetraS”, respectively), it can be noted that they are not usually detected in natural extractive CS samples whereas they typically characterise synthetic CS; Di-2,4,6triS is taken as a standard in order to evaluate the presence of triS CS in synthetic CS products as the other theoretically possible triS forms are not present in the naturally derived products.
The following Table 1 illustrates the main disaccharides identified in natural CS samples extracted and purified from various organs and tissues, mainly cartilages.
TABLE 1BovinePorcineChickenSharkRaySquidCSCSCSCSCSCSMn (kDa)12-17 9-14 8-1325-4027-3460-80Mw (kDa)20-2614-2016-2150-7050-70 80-120Polydispersity1.8-2.21.4-1.81.6-2.01.0-2.01.2-2.50.8-1.3IndexDi-0S 6 6 83313Di-6S (C)331420443915Di-4S (A)618072324350Di-2,6diS (D)NDNDND18130Di-4,6diS (E)NDNDND2122Di-2,4diS (B)NDNDND110triSNDNDNDNDNDNDtetraSNDNDNDNDNDNDCharge0.90-0.960.92-0.960.90-0.941.15-1.251.08-1.201.00-1.20Density4S/6S ratio1.50-2.004.50-7.003.00-4.000.45-0.901.00-1.402.50-4.00Mn = number average molecular weight;Mw = weight average molecular weight;Polydispersity Index = Mw/Mn;Charge Density is the number of sulfate groups per disaccharide units);ND = Not Detected
Table 1 illustrates the main structural parameters for the characterization of the principal natural CS samples purified from several sources.
In particular, molecular mass parameters are quite similar for terrestrial CS samples (bovine, porcine and chicken samples) but quite different from ichthyic samples (shark, ray and squid samples), the latter ones having molecular mass values greater than the former ones.
Furthermore, ichthyic CS samples have peculiar charge density values, greater than about 1.0, due to the presence of disulphated disaccharides and different from terrestrial samples having charge density values lower than about 1.0, due to the absence of disulphated disaccharides.
A further peculiarity of all natural CS is that when digested with chondroitinase ABC, a hydrolytic enzyme specific for either 4S or 6S sulphated disaccharides, as well as for unsulphated disaccharides, the polysaccharide chain is completely digested into disaccharide units. This can be easily observed with FACE (Fluorophore-Assisted Carbohydrate Electrophoresis) analysis. The complete digestion of natural CS is due to the absence of tri- and tetra-sulphated structures in the polysaccharide chain. Tri- and tetra-sulphated disaccharides, if present, are not recognised by chondroitinase ABC, not allowing a complete polysaccharide digestion, this produces partially undigested oligosaccharide chains easily determined in FACE analysis.
Finally, due to biosynthetic pathways, all known natural CS show the contemporary presence of disaccharides monosulphated in position 4 and position 6 of GalNAc (with the 4-sulphated disaccharide never lower than 30%), even if their ratio changes depending on the source.
As illustrated above, CS is a very complex heterogeneous macromolecule having variable structure and properties, depending on the extraction source.
Furthermore, as a result of the biosynthetic processes related to specific tissues and species, CS with different grades of polymerization may be biosynthesized producing macromolecules having various molecular masses and polydispersity.
Due to these structural variations, and in addition to the possible presence of specific oligosaccharide sequences, and purity of the preparations for therapy applications or in nutraceuticals, CS may have different properties and capacities.
In fact, different and peculiar activities have been reported depending on the CS structure [see, Volpi, N., Biomaterials 23, 3015, 2002; Volpi, N. et al., Biochimie 81, 955, 1999; Volpi, N., Biomaterials 20, 1359, 1999; and Suzuki, S. et al., J Biol Chem 243, 7, 1968].
Natural extractive CS is currently recommended by European League Against Rheumatism (EULAR) as a Symptomatic Slow Acting Drug for Osteo Arthritis (SYSADOA) in Europe in the treatment of knee OA [Jordan, K M et al., Ann Rheum Dis 62, 1145, 2003], hip [Jordan, K M et al., Ann Rheum Dis 62, 1145, 2003] and hand [Zhang, W. et al., Ann Rheum Dis 66, 377, 2007] based on research evidence and meta-analysis of numerous clinical studies.
Moreover, CS alone or in combination with other ingredients, is largely used as a nutraceutical, mostly in Europe and the United States [McAlindon, T E et al., JAMA 283, 1469, 2000. Volpi, N. et al., Food Anal Meth 1, 195, 2008. Volpi, N. et al., Separation Sc 1, 22, 2009].
CS effectiveness is strictly related to its anti-inflammatory activity, such as its ability to inhibit the activity of degradative enzymes as human leukocyte elastase (HLE) [Ronca, F. et al., Osteoarthritis Cartilage 6 Suppl A, 14, 1998. Egea, J. et al., Osteoarthritis Cartilage 18 Suppl 1, S24, 2010].
CS used worldwide in pharmaceutical or nutraceutical applications is obtained by extraction from tissues of several animals such as bovine and porcine [Fuentes, E P et al., Acta Farm Bonaerense 17, 135, 1998], avian [Luo, X M et al., Poult Sci 81, 1086-1089, 2002], cartilaginous fishes [Sugahara, K. et al., Eur J Biochem 239, 871, 1996. Lignot, B et al., J Biotechnol 103, 281, 2003], etc.
Yet, the animal origin of these products poses potential consumer safety problems associated with the possible presence of transmissible infective agents such as those causing spongiform encephalopathies in bovines, or due to a restriction in use related to religious issues.
In addition, the extractive nature of these products makes their supply potentially unreliable in view of a growing demand and increasing market volumes.
Such considerations have prompted the search for alternative, more dependable sources of CS, an example of which is the biotechnological production starting from the K4 capsular polysaccharide of E. coli as described in scientific and patent literature.
In this context, the term “biotechnological production” as used herein refers to a production method where a generally substantial portion of the final product is produced by a microorganism, or by isolated cells of a higher organism, in an artificial cultivation system, commonly and loosely referred to as “fermentation.”
Basically, three major biotechnological production approaches have been used so far in the art.
The first one can be identified with the production of CS-like compounds using as the starting material the K4 capsular polysaccharide of E. coli O5:K4:H4, which is then subjected to chemical transformation, whereas the second approach can be seen as the direct biosynthesis of CS-like compounds by microorganisms and the third one recognised to be the biosynthetic production of unsulphated chondroitin followed by chemical or biochemical sulphation.
EP-A-1304338, belonging to the above first approach, describes the production of CS starting from the K4 polysaccharide produced in liquid cultures that is first extracted and purified, and subsequently re-dissolved and subjected to acid hydrolysis, the main effect of which is the removal of the fructose residues linked to the GlcA residues present in the linear polymer. A secondary effect is the partial hydrolysis of the polysaccharide chain, leading to lower molecular mass products. Subsequently, the de-fructosylated polymer that is identical to unsulphated chondroitin is variously sulphated at the C-4 or at the C-6 positions of the GalNAc residues by chemical means using appropriate protective groups at the 4 or 6 positions. Also, a CS is therein disclosed, at least 70% of its content consisting of mono- and/or di-sulphated in the 4 and 6 positions of the galactosamine moiety, the 2 position of the glucuronic moiety being unsulphated, having a Mw of 6-25 kDa and a carboxyl/sulphate group ratio (i.e. charge density) of 0.7-2.0.
WO 2009/149155, exemplifying the above second approach, describes the direct production of CS-like compounds by several microorganisms, both bacteria and fungi. A CS terrestrial-like compound is also therein disclosed, both the 4- and 6-positions of the galactosamine moiety being sulphated; the compound is reported to show a molecular weight (Mw) from about 300 Da to 35 kDa and a 4S/6S sulphate ratio ranging from lower than 1 to higher than 1.
The third of the above approaches includes several different strategies for the production of unsulphated chondroitin, the main of which are the enzymatic synthesis of the polymer in cell-free systems, like the ones disclosed for instance in EP-A-1950308 and EP-A-1964924, and the biosynthesis in recombinant cells obtained expressing into hosts capable of producing, from UDP-GlcA, the genes kfoA and kfoC extracted from E. coli K4 described, for instance, in WO 2008/133350.
Another example of biosynthetic production of unsulphated chondroitin is disclosed by the Italian patent application No. MI2010A001300 which, inter alia, relates to a method for the biotechnological production of chondroitin comprising cultivating in a suitable medium a recombinant microorganism, preferably Escherichia coli DSM23644, recovering and purifying the unsulphated chondroitin present in the microbial culture and subsequently chemically sulphating the latter.
A common feature of the processes for the production of CS described so far is a substantial reduction of the molecular mass of the original material both during the acid-catalyzed removal of the fructose residues and during the chemical synthesis steps required for the sulphation of GalNAc residues.
As an example, EP-A-1304338 describes a 6-25 kDa molecular mass CS while the molecular mass of the K4 polysaccharide, used as the starting material, is disclosed to be 150-400 kDa.